All protein-coding genes in the protozoan parasite Trypanosoma brucei are arranged in long clusters and are transcribed into polycistronic precursor RNAs. These pre-mRNAs require further processing by coupled trans splicing and polyadenylation to generate mature mRNAs. Although studies in the last decade have identified numerous spliceosomal components, we still know very little about the components, mechanisms, and dynamics of the 3´ end-processing machinery in trypanosomes. Moreover, most genes in trypanosomes contain one to three trans splice and even more dispersed polyadenylation sites, indicating that the regulation of both processes provides another, still not very well explored level of post-transcriptional gene regulation in trypanosomes: In terms of polyadenylation, factors regulating the polyadenylation efficiency of a primary transcript with a single poly(A) site directly affect protein expression, since unprocessed transcripts are degraded or not exported to the cytoplasm. In addition, multiple polyadenylation sites allow the generation of different transcript isoforms of a single gene by alternative polyadenylation.To characterize the catalytic core of the polyadenylation complex in T. brucei, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3´ end processing of mRNAs. Based on tandem affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both in vivo polyadenylation and trans splicing, arguing for a general coupling of these two mRNA-processing reactions.By combining genome-wide analysis of expression (RNA-seq) and in vivo RNA binding (iCLIP), we identified for the first time a trans-acting RNA-binding protein, the trypanosomatid polypyrimidine tract binding protein (PTB/hnRNP I) homolog DRBD4, as a regulator of polyadenylation. Based on SELEX-seq and iCLIP, we delineated purine-rich sequences containing AUGA elements as DRBD4 RNA-binding motif and mapped in vivo binding sites mainly in untranslated regions (UTRs). Integrating RNA-seq and iCLIP datasets revealed that DRBD4 binds upstream of poly(A) sites and modulates both their activation and repression, thereby affecting general transcript and isoform expression levels.
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